The initiation of chromosome replication is the key to the control of chromosome replication and cell proliferation. We propose to work on the mechanism of the initiation of bacterial chromosomal and plasmid replication at their origins using Bacillus subtilis, and plasmids which can replicate in B. subtilis. Particularly interesting is our finding, by using temperature sensitive initiation mutants, that the replication origin of the chromosome is associated with the cell membrane, and that this membrane association is necessary for initiation. The isolation of the DNA-membrane complex from the mutants permitted us to demonstrate specific dissociation from the membrane of the host origin-carrying and plasmid DNA at high temperature (45 C), both in vivo and in vitro. Recently we found that a plasmid, pUB110, can bind to the membrane of B. subtilis in vitro, and that it has four membrane-binding areas in its DNA (4.5Kb). We have previously shown that pUB110 cannot initiate replication in one of the B. subtilis initiation mutants, dna-1 (a temperature sensitive mutant of the dnaB1 locus), at the non-permissive temperature, thus sharing a common initiation mechanism with the B. subtilis chromosome. This in vitro binding, type-II, is different from in vivo binding, type-I, in that the type-I binding is high-salt resistant and dependent on the product of a host initiation gene, dnaB1, whereas the type-II binding is high-salt sensitive and independent of the dnaB1 product. We will work on the molecular biology of these two types of binding, using plasmids and chromosomal origin. Specific points of emphasis will be: (1) further characterization of the type-I and type-II complexes, particularly the location and nucleotide sequence of the binding sites of pUB110 and the chromosomal origin; (2) development of an in vitro initiation system using the pUB110-membrane complex as the starting material; (3) determination of the binding location within the membrane.